The Adenylate Cyclase (CyaA) of Bordetella types in particular of Bordetella pertussis, has been described extensively as a recombinant vector able to deliver efficiently polypeptides, such as antigens, into the cytosol of antigen-presenting cells (APC) [1], [2], [3]. More, recombinant CyaAs have been used to efficaciously treat mice that bear tumours [4], [5], [6].
Several authors have highlighted that the efficiency of polypeptide-delivery, in particular antigen-delivery, by CyaA (used as a vector) can be positively or negatively affected by electrostatic charge of the inserted polypeptide (antigen) and its conformation. In 1998, Karimova & al. [7] described that the delivery of CD8+ T-cell polypeptide epitopes, inserted in CyaA, to and into antigen-presenting cells was dependent on the electrostatic charge of the inserted epitopes: a recombinant CyaA harbouring the OVA epitope was able to translocate into APC and to induce a CTL response in vivo, while the same construct with 4 glutamic residues fused to the OVA epitope, could no more translocate, and did not induce a detectable Cytotoxic T-cell Lymphocyte (CTL) response in vivo. In 2012, Holubova et al. described various constructs based on CyaA: either CyaA proteins deleted within their N-terminal comprising the OVA epitope SIINFEKL, or CyaA proteins truncated for their N-terminal domain and comprising several epitopes inserted at different sites [25]. Holubova et al. concludes that their experiments provide a proof of concept for construction of CyaA-based antigen delivery having the entire AC domain replaced by large artificial CTL polyepitope.
In 2001, Gmira & al. [8] developed a new CyaA vector to facilitate construction of recombinant CyaAs with exogenous polypeptides or antigens inserted within their catalytic domains. These modifications were:                the insertion of a multi-cloning site sequence with new unique restriction sites downstream from codon 224;        the deletion of codons 225 to 234; and        the change of codons 236, 238 and 239; these modifications were introduced to increase the local electrostatic charge (less acidic), which was previously shown to be critical for translocation of this CyaA-antigen hybrid protein across the cellular membrane of in situ APC.        
The modified CyaA had similar invasive activity as the wild type CyaA.
The authors have tested 5 antigens, whose size range from 87 to 206 residues, with an electrostatic charge from −4 to +14, and showed that those having an acidic value had lost their translocation efficiency, confirming previous results from Karimova et al. Moreover, they tested CyaAs with antigens with internal disulphide bridges or complex structures: none was able to translocate into the targeted cells, supporting the hypothesis that the polypeptides inserted in the catalytic domain of CyaA must unfold in order to be translocated.
TABLE 1extracted from [7] and [8]. Inserts with an acidic charge are nottranslocated into the cytosol of APCs. The acidic charge is calculated fromthe number of Lys and Arg residues minus the number of Asp and Glu residues.AntigenAntigenelectrostaticsizecharge (R/K-ActivityReferencesrCyaAantigen name/origin(aa)D/E)(+ or −)GmiraCyaANoneNANA+et al., 2001CyaA-NeuroBovine neurocalcine δ192−6−CyaA-RestAspergillus restrictus1485+restrictocinCyaA-DHFRMouse Dihydrofolate1877+reductaseKarimovaCyaA-TatTat HIV8714+et al., 1998CyaA-NefNef HIV206−4−CyaA-Ova21Ova class I epitope80+CyaA-Ova21-4EOva class I epitope + 412−4−glutamic acids
The antigens used in the case of tumour regression assays had either a short size (OVA is 8 residues in length) [4] or their secondary structure was disrupted by internal rearrangements of antigen segments and with a maximal size of 103 residues [5].
From these studies, the following conclusions to improve efficiency of vectorisation by the CyaA vector have been drawn:                Inclusion of acid regions in a polypeptide to be inserted into CyaA should be avoided; and        Inclusion of secondary and tertiary structures in these inserts should be avoided because such structures interfere with proper internalization of the enzymatic adenylate cyclase (AC) domain in which the polypeptide has been inserted.        
In view of these conclusions, two recombinant CyaAs were constructed one containing the HPV16 E7 antigen and the other one the HPV18 E7 antigen. In addition a bivalent recombinant CyaA was also constructed in which the HPV16 and HPV18 E7 antigens have been inserted together (patent EP1576967; Préville et al.). However, no assay was reported with a recombinant CyaA into which more than 2 HPV E7 proteins have been inserted into the same CyaA vector.
Thus, Préville and al. discloses three recombinant CyaA vectors into which the E7 polypeptide of the HPV16 type or variants thereof has been inserted.                the CyaA-E7full vector, containing the E7 protein full length,        the CyaA-E7Δ30-42 vector, containing E7 fragments deleted from the acidic domain from aa 30 to 42        the CyaA-E749-57 vector, containing a murine H-2Db-restricted T cell epitope present on E7.        
These recombinant CyaA vectors were used to immunize mice and to detect E7-specific CTL responses. To measure the immune response after mice immunisation, CTL Chrome Release assays were performed. In in vivo animal experiments, CyaA-E7Δ30-42 and CyaA-E7full gave the most efficient CTL immune response compared to CyaA-E749-57 
The ability of these recombinant CyaA vectors to induce tumour regression was also evaluated. If the rate of tumour regression conferred by CyaA-E749-57 and CyaA-E7full could not be noticeably differentiated, CyaA-E7Δ30-42 was clearly superior in terms of tumour regression and growth inhibition. Thus, the single CTL epitope previously shown to be recognized in C57BL/6 mice has been proved to be efficient, but did not give the most optimal immune response.
The persistence of the immune response was then tested. Splenocytes from some surviving mice after 3 months were tested for their ability to lyse TC-1 cells expressing the E7 antigen and the other surviving animals were re-challenged with TC-1 cells at day 100 post vaccination. Animals vaccinated with CyaA-E7Δ30-42 displayed a high level of protection. Less than 40% of animals vaccinated with CyaA-E749-57 were protected while 90% to 100% of animals vaccinated with CyaA-E7Δ30-42 and CyaA-E7full survived.
The following teachings can be extracted from this work:                CyaA vectors carrying HPV16 and/or HPV18 E7 proteins lead to an immune response in C57BL/6 mice;        a complete response is obtained with an antigen having both its CD8+ and CD4+ T cell epitopes, as compared to the E749-57 epitope which has a CD8+ T cell epitope only;        a superior efficiency is obtained in mice treated with the CyaA-E7Δ30-42 vector where the E7 protein is deleted of its acidic region from residues 30 to 42, as compared to mice treated with CyaA-E7full or CyaA-E749-57;        the immune response obtained with these vectors is able to induce regression of tumour lesions;        a long lasting response is obtained, since a new challenge with TC-1 cells, in treated tumour free mice, is rejected; and        the co-injection of two recombinant CyaAs in order to develop a bivalent therapy is possible, each antigen keeping the response against its epitopes respectively.        
Therefore, in the prior art, acidic amino acid stretches embedded in certain polypeptides or antigens and overall negatively charged polypeptides or antigens have been shown to alter the efficiency of a CyaA vector to translocate these polypeptides, across the cell membrane of APC in vaccinated animals. This leads to weak or no protective cellular immune responses against said antigens.
The inventors consider that this may be regarded as a drawback for the design of drug candidates, since such acidic amino acid sequences may contain important CD4+ epitopes and/or CD8+ epitopes, required for protective cellular immunity.
Therefore, there is still a need for improved vectors bearing immunogenic constructions which can be used to induce strong and long lasting cellular protective immune responses, in particular in tumour regression and tumour prevention, against polypeptides and antigens encompassing acidic amino acid stretches and against overall negatively charged polypeptides or antigen.